CONSTRUCTION OF SINGLE AMINO-ACID SUBSTITUTION MUTANTS OF CLONED BACILLUS-STEAROTHERMOPHILUS DNA-POLYMERASE-I WHICH LACK 5'-]3'-EXONUCLEASEACTIVITY

Two individual amino acid substitutions were engineered at a selected site in the 5' --> 3' exonuclease domain of the cloned Bacillus stearo thermophilus DNA polymerase I gene. These mutations resulted in the ex pression of enzymes lacking the 5' --> 3' exonuclease activity while m aintaining normal polymerizing activity. The mutated and non-mutated e nzymes were each constitutively expressed in an Escherichia coli host without the use of an exogenous or inducible promoter, and the mutated enzymes were demonstrated to be equivalent to the subtilisin large fr agment of the native holoenzyme in sequencing reactions.